Streptavidin (“SA”) is a 159 amino acid protein produced by Streptomyces avidinii, and which specifically binds water-soluble biotin (Chaiet et al., Arch. Biochem. Biophys. 106:1–5, 1964). Streptavidin is a nearly neutral 64,000 dalton tetrameric protein. Accordingly, it consists of four identical subunits each having an approximate molecular mass of 16,000 daltons (Sano and Cantor, Proc. Natl. Acad. Sci. USA 87:142–146, 1990). Streptavidin shares some common characteristics with avidin, such as molecular weight, subunit composition, and capacity to bind biotin with high affinity (KD≈10−15) (Green, Adv. Prot. Chem. 29:85–133, 1975). Further, while streptavidin and avidin differ in their amino acid compositions, both have an unusually high content of threonine and tryptophan. In addition, streptavidin differs from avidin in that it is much more specific for biotin at physiological pH, likely due to the absence of carbohydrates on streptavidin. Various comparative properties and isolation of avidin and streptavidin are described by Green et al., Methods in Enzymology 184:51–67, 1990 and Bayer et al., Methods in Enzymology 184:80–89, 1990.
The streptavidin gene has been cloned and expressed in E. coli (Sano and Cantor, Proc. Natl. Acad. Sci. USA 87(1):142–146, 1990; Agarana, et al., Nucleic Acids Res. 14(4):1871–1882, 1986). Fusion constructs of streptavidin, and truncated forms thereof, with various proteins, including single-chain antibodies, have also been expressed in E. coli (Sano and Cantor, Biotechnology (NY) 9(12):1378–1381, 1991; Sano and Cantor, Biochem. Biophys. Res. Commun. 176(2):571–577, 1991; Sano, et al., Proc. Natl. Acad. Sci. USA 89(5):1534–1538, 1992; Walsh and Swaisgood, Biotech. Bioeng. 44:1348–1354, 1994; Le, et al., Enzyme Microb. Technol. 16(6):496–500, 1994; Dubel, et al., J. Immunol. Methods 178(2):201–209, 1995; Kipriyanov, et al., Hum. Antibodies Hybridomas 6(3):93–101, 1995; Kipriyanov, et al., Protein Eng. 9(2):203–211, 1996; Ohno, et al., Biochem. Mol. Med. 58(2):227–233, 1996; Ohno and Meruelo, DNA Cell Biol. 15(5):401–406, 1996; Pearce, et al. Biochem. Mol. Biol. Int. 42(6):1179–1188, 1997; Koo, et al., Applied Environ. Microbiol. 64(7):2497–2502, 1998) and in other organisms (Karp, et al., Biotechniques 20(3):452–459, 1996). Sano and Cantor (PNAS, supra) found that expression of full-length forms of streptavidin was lethal to E. coli host cells and, when capable of being expressed in truncated forms (e.g., under a T7 promoter system), only poor and varied expression was observed and the protein remained in inclusion bodies. However, there are also published reports of the expression of soluble streptavidin in E. coli (Gallizia et al., Protein Expr. Purif. 14(2):192–196, 1998; Veiko et al., Bioorg. Khim. 25(3):184–188, 1999). Those of skill in the art have frequently used “core streptavidin” (residues 14–136), or similar truncated forms, in the preparation of fusion constructs. The basis of the use of core residues 14–136 has been the observation that streptavidin preparations purified from the culture medium of S. avidinii have usually undergone proteolysis at both the N- and C-termini to produce this core structure, or functional forms thereof (Argarana et al., supra).
Presently, preparations of streptavidin expressed gene fusions are usually made by expressing a core streptavidin-containing construct in bacteria, wherein inclusion bodies are formed. Such production has several disadvantages, including the rigor and expense of purifying from inclusion bodies, the necessity of using harsh denaturing agents such as guanidine hydrochloride, and the difficulty in scaling up in an economical fashion. To a lesser extent, there has also been reported periplasmic expression of core streptavidin-containing constructs in soluble form (Dubel et al., supra).
Therefore, there exists a need in the art for easy, cost effective, and scaleable methods for the production of streptavidin fusion proteins. Accordingly, the present invention provides several key advantages. For example, in one embodiment, a genomic streptavidin expressed gene fusion is expressed as a soluble protein into the periplasmic space of bacteria and undergoes spontaneous folding. Accordingly, such expression offers the advantage that the periplasm is a low biotin environment and one need not purify and refold the protein under harsh denaturing conditions that may prove fatal to the polypeptide encoded by a heterologous nucleic acid molecule fused to the genomic streptavidin nucleic acid molecule. The present invention fulfills this need, while further providing other related advantages.